An Accurate Prediction of the pH Change Due to Degradation: Correction for a “Produced” Secondary Buffering System

Esmolol hydrochloride degrades in aqueous solutions by the hydrolysis of a labile aliphatic carboxy-ester group. The products are methanol and ASL-8123. The resulting aliphatic carboxylic acid moiety (ASL-8123) has a pK of 4.80, which is within 1 pH unit of the pH of the formulation. ASL-8123 therefore acts as a secondary buffer and minimizes the change in pH due to degradation. Equations are presented to calculate the change in the pH when the primary degradation product acts as a secondary buffer. This information can be used in the development of a parenteral product to predict, a priori, the concentration of buffer necessary for optimal pH maintenance. This knowledge can reduce the number of formulation screens required to determine the necessary buffer capacity for optimal drug stability.     

Guided tissue regeneration in the treatment of furcation defects in mandibular molars

The present investigation was designed to evaluate the regenerative potential of the periodontal tissues in degree III furcation defects at mandibular molars using a treatment procedure based on the principle of guided tissue regeneration. The patient sample included 21 patients, 26-65 years of age, who presented periodontal lesions in the right and left molar regions including "through and through" furcation defects. After an initial examination, each patient was subjected to a series of full-mouth scaling and root planing. 2-3 months later, they were recalled for a baseline examination. The furcation-involved molars were randomly assigned in each patient to either a test or a control treatment procedure. The test procedure included the elevation of muco-periosteal flaps at the buccal and lingual aspects of the molars. Granulation tissue was removed and the exposed root surfaces were debrided and planed. The width and the height of the entrance openings to the furcation defects were assessed. A teflon membrane was adjusted to cover the entrances to the defects (buccal and lingual) and was retained in the manner described by Pontoriero et al. (1988). The flaps were repositioned on the outer surface of the membrane and secured by sutures which were removed after 10 days. Following surgery, the patients were instructed to rinse the mouth twice daily for 4 weeks with chlorhexidine gluconate. The membranes were removed after a healing period of 1-2 months. A surgical procedure identical to the test procedure was performed in the control tooth regions with the exception of the placement of membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Retrospectroscope: The single breath test of diffusing capacity for carbon monoxide

In this paper I briefly review the significance of the single breath test of diffusing capacity for carbon monoxide (D_LCO). I then attempt to answer the question of why this simple test has not become widely used in the past twenty years. The reasons are primarily due to a failure in communication between research workers and practicing physicians. The practicing clinicians have difficulty with mathematical analysis and do not appreciate the limits for which the test is valid. Under steady-state conditions D_LCO measures the transfer factor for the whole lungs, i.e. the volume of CO taken up by pulmonary capillary blood from alveolar gas when the alveolar CO pressure is one. It is independent of regional inhomogeneities. D_L is the product of Krogh's K and alveolar volume. Krogh's K tends to become constant near total lung capacity while D_L does not. For this reason K will probably vary less than D_L and should always be reported when doing this test. In terms of the simple lung model with a single homogeneous alveolar compartment K is equal to D_L per unit alveolar volume. I expect the measurement of K and D_LCO by the modified Krogh single breath test to become a screening test for lung disease in its early stages.     

CD4 T cells in tumor immunity

T cell immunity is the key to protective immune responses against tumors. Traditionally, this function has been ascribed to CD8 T lymphocytes with cytotoxic activity, which are restricted by MHC class I molecules. In recent years the realization that CD4 T cells can also play a relevant role in protective anti-tumor responses has received growing attention. Here we will discuss the role of MHC class II-restricted T cells in response to, and in the regulation of, tumor antigens. Emphasis will be placed on four areas: (1) the role of CD4 T cell immunity in tumor protection in animal models and putative mode of action, (2) tumor antigens recognized by human CD4 T cells, (3) the cooperation between two CD4 T cells of different specificity as a new way to jump start the response against sub-immunogenic determinants of tumor antigens in a tolerant environment, and (4) the negative impact of regulatory CD4 T cells on anti-tumor T cell responses. By drawing attention to these four areas, it is our intention to provide the reader with a comprehensive view of issues of contemporary importance for this field, in the expectation that the information will help a better design of therapeutic cancer vaccines.     

High-throughput detection of pathogenic yeasts of the genus trichosporon

The need for a rapid and accurate method for the detection of fungal pathogens has become imperative as the incidence of fungal infections has increased dramatically. Herein, we tested the Luminex 100, a novel flow cytometer, for the detection of the medically important genus Trichosporon. This genus was selected as our proof-of-concept model due to the close phylogenetic relationship between the species. The method, which is based on a nucleotide hybridization assay, consists of a combination of different sets of fluorescent beads covalently bound to species-specific capture probes. Upon hybridization, the beads bearing the target amplicons are classified by their spectral addresses with a 635-nm laser. Quantitation of the hybridized biotinylated amplicon is based on fluorescence detection with a 532-nm laser. We tested in various multiplex formats 48 species-specific and group-specific capture probes designed in the D1/D2 region of ribosomal DNA, internal transcribed spacer regions, and intergenic spacer region. Species-specific biotinylated amplicons were generated with three sets of primers to yield fragments from the three regions. The assay was specific and fast, as it discriminated species differing by 1 nucleotide and required less than 50 min following amplification to process a 96-well plate. The sensitivity of the assay allowed the detection of 10(2) genome molecules in PCRs and 10(7) to 10(8) molecules of biotinylated amplification product. This technology provided a rapid means of detection of Trichosporon species with the flexibility to identify species in a multiplex format by combining different sets of beads.     

Angiogenic response induced by acellular aortic matrix in vivo

In this study, we investigated the angiogenic response induced by acellular aortic matrices implanted in vivo onto the chick embryo chorioallantoic membrane (CAM), a useful model for such investigation. Results showed that acellular matrices were able to induce a strong angiogenic response comparable to that of fibroblast growth factor 2 (FGF-2), a well-known angiogenic cytokine. The angiogenic response was further increased when exogenous FGF-2 or transforming growth factor beta 1 (TGF-beta1) were added to the matrices and inhibited by the addition of an anti-FGF-2 or anti-TGF-beta1 antibodies. The response may be considered dependent on a direct angiogenic effect exerted by the matrices and in part also by the presence of FGF-2 and TGF-beta1 in the acellular matrices.     

Human ORFeome Version 1.1: A Platform for Reverse Proteomics

The advent of systems biology necessitates the cloning of nearly entire sets of protein-encoding open reading frames (ORFs), or ORFeomes, to allow functional studies of the corresponding proteomes. Here, we describe the generation of a first version of the human ORFeome using a newly improved Gateway recombinational cloning approach. Using the Mammalian Gene Collection (MGC) resource as a starting point, we report the successful cloning of 8076 human ORFs, representing at least 7263 human genes, as mini-pools of PCR-amplified products. These were assembled into the human ORFeome version 1.1 (hORFeome v1.1) collection. After assessing the overall quality of this version, we describe the use of hORFeome v1.1 for heterologous protein expression in two different expression systems at proteome scale. The hORFeome v1.1 represents a central resource for the cloning of large sets of human ORFs in various settings for functional proteomics of many types, and will serve as the foundation for subsequent improved versions of the human ORFeome.     

Comparison of sensitivity for human cytomegalovirus of the polymerase chain reaction, traditional tube culture and shell vial assay by sequential dilutions of infected cell lines.

Although traditional tube culture (TTC) is still considered by many as the 'gold standard' for the laboratory diagnosis of human cytomegalovirus (HCMV), the shell vial assay (SVA) offers greater speed of detection. This technique utilizes immunofluorescence (IF) to detect early or immediate early nuclear antigens (IEA). The detection capabilities of these two tests were compared with the polymerase chain reaction (PCR), a technique that amplifies enzymatically selected DNA target sequences. Serial dilutions of crude culture harvests from 2 HCMV strains, Towne and a clinical urine isolate, were made up to 1:1 000,000. Ten-microliters aliquots of the original sample and each dilution were tested by PCR, TTC and SVA. For PCR, the nested-primer approach was used. Outer primers delimited a 721-bp sequence contained within the 2nd to 4th exons of the immediate-early protein. Inner nest primers delimited a 167-bp sequence in the third exon, detected by a 32P-labelled probe. The results show that: (1) control samples which contained all PCR reagents but no DNA were uniformly negative; (2) radiolabelled-probe detection (RPD) of PCR products is, on average, 100 x more sensitive than detection by ethidium bromide; (3) PCR is, on average, 100 x more sensitive than evaluation of cytopathic effect (CPE) in the TTC; (4) the predictive value of a negative SVA result is low compared to PCR.